Mutation screening
We obtained DNA samples for each patient from peripheral blood
lymphocytes by standard procedures (Sambrook, Russell, & Fritsch,
2001). All 11 coding exons and intron-exon boundaries of EFHC1were amplified by polymerase chain reaction (PCR; primer sequences
available upon request). Sanger sequencing was performed by capillary
electrophoresis in an ABI 3500xL Genetic Analyzer using the BigDye®
Terminator Cycle Sequencing Kit (Thermo Fisher Scientific, Waltham, MA,
USA). Sequence variants were described according to the conventional
nomenclature (den Dunnen & Antonarakis, 2001; den Dunnen, 2019) based
on the full-length EFHC1 isoform (NM_018100) and deposited in a
public genomic database of GGEs (http://bipmed.iqm.unicamp.br/GGE).